Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 are an important cause of food-borne outbreaks of bloody diarrhea and the potentially fatal hemolytic uremic syndrome. EHEC infection is characterized by the formation of attaching and effacing (A/E) lesions on the intestinal epithelium. A type III secretion system (T3SS) responsible for this histopathology is encoded by the locus of enterocyte effacement (LEE) pathogenicity island. The ability of EHEC to co-ordinate the expression of virulence factors in response to changing environments is crucial for its survival and successful colonization of the host, which is why understanding of the regulatory basis for virulence is important for identifying new therapeutic strategies. The expression of virulence genes including those of the LEE is tightly regulated. While protein phosphorylation of response regulators on histidine is well-known to regulate virulence gene expression, the regulatory role of protein tyrosine phosphorylation in bacteria is poorly understood, especially considering the prominent role of phosphotyrosine-mediated signaling in eukaryotes. Using a highly sensitive mass spectrometry-based phosphoproteomics approach, we recently found that the number of tyrosine-phosphorylated proteins in E. coli was about 10-fold higher than previously reported. The newly identified proteins relate to fundamental cell functions and virulence, indicating a central regulatory role of tyrosine phosphorylation. The proposed study aims to further elucidate the regulatory role of phosphotyrosine-mediated signaling in EHEC virulence with the focus being on Cra, which was among proteins identified as tyrosine phosphorylated. Cra, a DNA-binding global glycolytic metabolite-responsive regulator controlling sugar metabolism, is required for LEE expression under gluconeogenic conditions to ensure successful colonization. Given our data indicating that phosphorylation of the functionally important Cra Y47 residue, located in the DNA-binding domain, negatively affects the production and secretion of T3SS proteins, we hypothesize that phosphorylation of Cra Y47 negatively controls expression of virulence genes by interfering with Cra DNA-binding capacity. We also hypothesize that Cra phosphorylation occurs in response to carbon source availability. The following Specific Aims address these hypotheses: Aim 1: i) Determine whether Cra tyrosine phosphorylation is induced by glycolytic conditions; and ii) Determine the global regulatory effect of phosphotyrosine-mediated regulation of Cra. Aim 2: i) Determine the role of tyrosine phosphorylation in Cra-mediated regulation of the T3SS including assessing the effect of Cra tyrosine phosphorylation on LEE expression and A/E lesion formation; and ii) Determine if phosphotyrosine-mediated regulation of Cra affects DNA-binding. To successfully address these aims, we will use innovative approaches including the RNA-Seq technology and a quantitative high resolution mass spectrometry approach. Increased knowledge on how tyrosine regulates EHEC virulence can facilitate the identification of novel therapeutic approaches to treat EHEC, for which antibiotic therapy is contraindicated.